The transmission of DNA polymers across biological membranes is a fundamental process in all cells. Early studies in bacteria demonstrated the remarkable capacity of a mobile genetic element (MGE), the conjugative F plasmid, to integrate into the genome and promote single-stranded transfer of the entire chromosome to recipient bacteria in uninterrupted matings. In the ensuing ~75 years, studies established the broad importance of MGEs in shaping of bacterial genomes over evolutionary time as well as their capacity to rapidly disseminate antibiotic resistance and other fitness traits under selective pressure. F plasmids are particularly problematic from a medical perspective because they are distributed among pathogenic members of the Enterobacteriaceae and they often harbor resistance to multiple antibiotics including last-resort ?-lactam antibiotics, e.g., carbapenems. F plasmids also have the capacity to transfer at very high frequencies and elaborate conjugative pili in large numbers around the cell surface; these attachment organelles play important roles in establishment of robust biofilms, which are typically refractory to antibiotic treatment. Bacterial MGEs, including the F plasmids, are transmitted intercellularly through nanomachines termed type IV secretion systems (T4SSs). F-encoded and phylogenetically-related conjugation systems have served as important models for deciphering the mechanism of action of T4SSs, but there remains a fundamental lack of structural detail for these machines. We have solved, for the first time, structures of F pilus-associated T4SS nanomachines in the native environment of the bacterial cell envelope by cryoelectron tomography (cryoET). Strikingly, we discovered that the F system elaborates four distinct F-substructures, not just one or two as previous models have predicted. These include a presumptive F channel complex and three distinct substructures associated with F pili. Our overarching hypothesis is that the F channel complex transitions to an active translocation channel or a pilus assembly factory (PAF) in response to distinct signals; the PAF in turn has the potential to produce many pili for deposition on other F-encoded platforms. This exploratory study will test this new model by solving WT and mutant T4SSs at the highest resolutions achievable using in situ cryoET. In Aim 1, we will generate 3D maps of WT and mutant substructures at resolutions sufficient to define the structural contributions of the F pilus and TraA pilin, as well as the TraC and TraD T4SS ATPases, to the visualized substructures. Aim 2 initiates a long-term goal of defining compositions and assembly pathways for the F-encoded substructures through analyses of tra gene deletion mutations and detection of novel densities contributed by traceable tags fused to Tra proteins for spatial assignments. Our studies will generate important new insights into the composition, architecture, and biogenesis of F T4SSs, and answer long-standing questions pertaining to the physical relationship of the T4SS and pilus. We fully anticipate our findings will lead to major paradigm shifts in this field, and set the stage for design of intervention therapies.